Collagen plays a central role as a biomaterial and as a scaffold in the regenerative tissue replacement strategies. Existing synthetic analogs of collagen have extremely poor biomechanical properties in comparison to the tissues they are targeted to replace due, in part, to the lack of orientation in hierarchical orders above the level of fibers. The overall goal of the proposed study is to demonstrate the feasibility of a novel biomechanically competent collagenous tissue engineering scaffold fabricated by: 1) an unconventional electrochemical process to attain an unprecedented level of molecular alignment and molecular packing density persistent across multiple levels of structural hierarchies, and, 2) the control of interfibrillar attachment by use of a biomimetic decorin-like linkage molecule. Phase 1 of proposed studies will elucidate the mechanisms by which collagen solutions achieve long-range order under the effect of weak currents applied to directly to the solutions. The effects of pH gradient electric current amplitude and collagen concentration on the hierarchical organization of collagen will be investigated to optimize the synthetic process. The strength of resulting oriented collagen gels will be improved by identifying the appropriate type and concentration of crosslinking amongst genipin and glutaraldehyde. {The D-banding pattern and collagen fibril diameter will be improved by modulating the phosphate buffered saline treatment conditions.} The viscoelastic properties of oriented constructs will be modified by decorin mimics consisting of dermatan sulfate attached to peptide motifs which selectively bind to type I collagen molecules. Mechanical properties of resulting synthetic constructs will be assessed by macroscale mechanical tests and compared to those of rat tendon, a reference natural tissue. {Phase 2 is going to assess the differentiation of bone marrow derived mesenchymal stem cell progenitors on this material towards tenocytic lineage and foster this differentiation pathway by the mechanical cue.} Overall, the proposed study will: a) optimize the fabrication process variables of a novel fabrication method to obtain highly oriented tendon-mimicking constructs, and, b) assess the feasibility of the material as a tissue engineering scaffold by investigating phenotypic and genotypic behavior of bone marrow derived stem-cell progenitors seeded within the three-dimensional structures consisting of multiple collagenous bundles. A biomaterial of this nature may play a key role in creating strategies towards replacement of load bearing connective tissues such as tendons, ligaments, bones and vascular walls provided that the current study proves its feasibility. PUBLIC HEALTH RELEVANCE: The overall goal of the proposed study is to demonstrate the feasibility of a novel collagenous tissue engineering scaffold with mechanical properties converging those of tendon or ligaments. The study will assess the promise of this novel material by investigating whether adult stem cells seeded on this material differentiate to act like tendon cells.